Association of ANRIL Expression with Coronary Artery
Disease in Type 2 Diabetic Patients
Materials and Methods
In this case-control study, we examined
Expression analysis revealed that
The expression level of
Long noncoding RNAs (lncRNAs) are one of the most
important classes of RNA molecules, receiving extensive
attention as potentially novel biological regulators. Manyroles have been attributed to lncRNAs including nuclear
organization, dosage compensation, epigenetic modificationand RNA splicing (1, 2). Accumulated evidence has shownthat lncRNAs can exert their regulatory function in both
cis and trans patterns (3). It has also been suggested thatderegulation of lncRNAs, as a key regulator of normal cellfunction, is correlated with different types of human disorders.
For example, the
Genome-wide association studies (GWAS) have
revealed that the 9p21 locus is associated with several
diseases, including CAD, T2DM and several types
of cancer (7). This locus overlaps with the well-
T2DM is a well-recognized cause of multiple complications including retinopathy, nephropathy and coronary artery disease (CAD) (12-14). Atherosclerosis is the leading cause of morbidity and mortality of T2DM patients. Prevention of CAD morbidity and mortality in patient with T2DM has therefore become a major health issue worldwide (15). Given that T2DM and atherosclerosis are two closely linked disorders, many efforts have been carried out to elucidate their common etiology. Risk factors including abdominal obesity, insulin resistance and inflammation are involved in these diseases (16, 17).
As a genomic hotspot of CAD and T2DM, we aimed to
examine the expression profile of
Materials and Methods
The subjects of this case-control study were 64 patients who had undergone coronary angiography at the Tehran Heart Center, Iran. Patients were screened for the presence of diabetes [fasting blood sugar (FBS)≥126 mg/dl (6.9 mmol/L) and/or HbA1c≥6.5%] and those who qualified as diabetic were included in the study. T2DM patients were then divided into two groups (33 CAD+ patients and 31 CAD- patients). According to the results of coronary angiography, diabetic patients with coronary artery stenosis (≥50%) were chosen as CAD+ and further classified into single-vessel disease (SVD, n=11) and multi-vessel disease (MVD, n=22) sub-groups. Also, high-density lipoprotein (HDL)cholesterol and triglyceride levels were assessed and low- density lipoprotein cholesterol level in plasma was measured by Friedewald’s formula. All subjects gave informed written consent to participate in the study. This study was approved by the Ethics Committees of Tehran Heart Center and Tarbiat Modares University.
Blood collection and peripheral blood mononuclear cells isolation
Whole blood was collected from patients on the day of coronary angiography. All patients were informed not to take any food and medication for at least 12 hours before blood collection. Peripheral blood mononuclear cells (PBMCs) were immediately isolated by centrifugation by the Ficoll-PaqueTM (lympholyte, Cedarlane, Netherlands) gradient according to the manufacturer’s instructions.
RNA extraction and cDNA synthesis
The acid guanidinium-phenol-chloroform method with the RNXTM-Plus reagent (SinaClon Co., Iran) was used to extract total RNA from isolated PBMCs. The integrity and quality of total RNA was assessed by agarose gel electrophoresis, and its concentration was examined by spectrophotometry at 260 nm. After treatment with DNase I (Fermentas, Lithuania), to eliminate DNAcontamination, 3 µg of total RNA was used to synthesize complementary DNA (cDNA) by using random hexamer and oligo (dT)18 primers along with the M-MulV reverse transcriptase (Thermo Scientific, USA) in a total reaction volume of 20 µl, according to the manufacturer’s instructions.
Quantitative real-time polymerase chain reaction
Quantitative real-time polymerase chain reaction (qPCR)
was undertaken in an ABI StepOne™ (Applied Biosystems,
Foster City, CA, USA) machine. The expression of
was used as an internal control. All of the samples were runin triplicate and the normalized expression levels were usedfor further analysis. The level of differential expression was calculated by the 2-ΔΔCt method (12).
Data were shown as mean ± SEM and analyzed fornormality with the Shapiro-Wilk test. Mann-Whitney U-testwas used to assess the statistical significance of the differentialgene expression between CAD+ and CADpatient groups.
Chi-square test, Student’s t test or Mann-Whitney U test wereperformed to compare demographic variables between CAD-
versus CAD+ patients Pearson correlation coefficient was usedassess the correlation of
ANRIL expression in the peripheral blood mononuclear
cells of patients
The expression of
Effect of glycemic control and lipid profile on the
expression level of
Next, we examined whether glycemic control or the
lipid profile of patients is related to the expression
ANRIL as a potential biomarker for progression of
atherosclerosis in T2DM
Receiver operating characteristic (ROC) curve analysis
was performed and the area under the ROC curve (AUC)
was calculated to examine whether
|Characteristic||CAD n=33 (100%)||non-CAD n=31 (100%)||P values|
|60.76 (9.093)||61.10 (8.047)||0.875**|
|29.19 (5.10)||27.94 (3.89)||0.554***|
|8.45 (1.84)||7.76 (1.27)||0.019***|
Data are mean ± SD or number of subjects (%). BMI; Body mass index, CAD; Coronary artery disease, HDL; High density lipoprotein, LDL; Low density lipoprotein, TCH; Total cholesterol, HbA1C; Glycated hemoglobin, *; Chi-square test, **; Student’s t test, and ***; Mann-Whitney U test were performed to compare variables between CADversus CAD+ patients.
|Correlation with||r*||P value|
*; Pearson correlation coefficient, HbA1C; Glycated hemoglobin, FBS; Fasting blood sugar, HDL; High density lipoprotein, LDL; Low density lipoprotein, and TCH; Total cholesterol.
Currently extensive research is undertaken regarding
lncRNA as potential biomarkers and has become one
of the most popular areas in molecular medicine.
Association of lncRNAs with inflammatory diseases,
such as atherosclerosis and T2DM, has been discovered
recently. The remarkable change in lncRNA expression
in inflammatory diseases such as CAD seems to be a
feature shared among some lncRNAs, rendering them
as potential biomarkers as well as therapeutic targets
(17, 18). However, only a few lncRNAs including the
metastasis-associated lung adenocarcinoma transcript
This up-regulation might be associated with the
progression of CAD in T2DM patients. Holdt et al. (10)
showed that expression of
What might be the role of
We show that the association of the 9p21 locus with
CAD in T2DM patients is likely to be due to
The authors gratefully acknowledge the contribution of the patients and the institutions in this study. The Iran National Science Foundation and the Department of Research Affairs of Tarbiat Modares University provided the funding of this work. The authors declare that there is no conflicts of interest.
E.R.; Participated in study design, data collection and evaluation and drafting. A.A.; Sample collection. M.A.B.; Participated in study design and sample collection. B.M.S.; Participated in study design. M.B.; Participated in study design, data collection and evaluation and responsible for overall supervision. All authors read and approved the final manuscript.